Isolated human phospholipase proteins, nucleic acid molecules encoding human phospholipase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the phospholipase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the phospholipase peptides, and methods of identifying modulators of the phospholipase peptides.

RELATED APPLICATIONS

The present application claims priority to PROVISIONAL APPLICATION U.S.Ser. No. 60/232,632, Filed Sep. 14, 2000.

FIELD OF THE INVENTION

The present invention is in the field of phospholipase proteins that arerelated to the phospholipase C subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins that effect protein phosphorylation and nucleicacid molecules encoding such peptide and protein molecules, all of whichare useful in the development of human therapeutics and diagnosticcompositions and methods.

BACKGROUND OF THE INVENTION

Phospholipases

There are three major families of known human phospholipase enzymes:Phospholipase A2, Phospholipase C, and Phospholipase D.

Enzymes in the Phospholipase A2 family (“P1A2”) hydrolyze the sn-2 fattyacid acyl ester bond of phosphoglycerides, releasing free fatty acidsand lysophospholipids. The PlA2s constitute a diverse family of enzymeswith respect to sequence, function, localization and divalent cationrequirements. They play an important role in a variety of cellularprocesses, including the digestion and metabolism of phospholipids aswell as the production of precursors for inflammatory reactions. ThePlA2s have been classified into at least 5 groups (although differentclassification schemes exist and up to 10 groups have been identified bysome authorities) based on their size, structure and need for divalentcations. Groups I, II and III all contain secreted forms of PlA, whichare extracellular enzymes that have a low molecular mass and requirecalcium ions for catalysis. Groups IV and V contain cytosolic forms ofPlA2s that have a high molecular mass and do not necessarily requirecalcium ions.

Amongst the best characterized of the PlA2 phospholipases are digestiveenzymes secreted as zymogens by the pancreas. These enzymes, which areinvolved in the hydrolysis of dietary phospholipids, have stronghomology to the venom phospholipases of snakes. Other PlA2s playimportant roles in the control of signaling cascades such as thecytosolic PlA2, Group IVA enzyme (“PLA2G4A”) which catalyzes the releaseof arachidonic acid from membrane phospholipids. Arachidonic acid servesas a precursor for a wide spectrum of biological effectors, collectivelyknown as eicosanoids (and including the prostaglandin group ofmolecules) that are involved in hemodynamic regulation, inflammatoryresponses and other cellular processes.

Another biologically active phospholipid, platelet-activating factor(“PAF”) is hydrolyzed to metabolically-inactive degradation products bythe group VII PlA2 known as PAF acetylhydrolase. Deficiency of PAFacetylhydrolase has been reported in patients with systemic lupuserythematosis and increased levels of PAF have been reported in childrenwith acute asthmatic attacks. Elevated levels of the group II PlA2 knownas PLA2G2A have been reported in plasma and synovial fluid in patientswith inflammatory arthritis. Studies of a mouse colon cancer modelshowed that alleles of the murine ortholog of this gene were able tomodify the number of tumors that developed in animals with multipleintestinal neoplasia (a mouse model of the human disorder known asfamilial adenomtous polyposis). Subsequent studies in humans showedmutations in PLA2G2A were associated with the risk of developingcolorectal cancer. PLA2G2A is presumed to act through altering cellularmicroenvironments within the intestinal crypts of the colonic mucosa,although the precise mechanism by which this effect is exerted is notclear.

Enzymes in the Phospholipase C (“PLC”) family catalyze the hydrolysis ofthe plasma membrane phospholipids, phosphatidyl inositol phosphate(“PIP”) or phosphatidylinositol 4,5-biphosphate (“PIP2”), generating asproducts the second messengers, 1,4,5-inositol triphosphate (“IP3”) and1,2-diacylglycerol (“DAG”). Molecules belonging to the PLC gene familyare divided into subfamilies, PLC-beta, PLC-gamma and PLC-delta.PLC-delta is distinguished from PLC-gamma by lack of the SH2 and SH3domains that are essential for activation of PLC-gamma by tyrosineprotein kinases. PLC-delta is distinguished from PLC-beta by lack of theC-terminal region of PLC-beta that is responsible for binding andactivation of G proteins. Various PLC enzymes play important roles insignal transduction cascades throughout the body. Activating signalsinclude hormones, growth factors and neurotransmitters. One of thefunctions of IP2 is to modulate intracellular calcium levels while DAGis involved in the activation of certain protein kinases and can promotemembrane fusion in processes involving vesicular trafficking.

The novel human protein provided by the present invention is related tothe phospholipase C family, and shows a particularly high degree ofsimilarity to enzymes of the phospholipase C-delta subclass. PLC-deltaproteins may be associated with abnormal calcium homeostasis andincreased intracellular calcium ion concentrations, conditions commonlyassociated with hypertension (Yagisawa et al, J Hypertens 1991Nov;9(11):997-1004). Furthermore, the gene encoding PLC-deltal islocated just distal to a region of chromosome 3 that is deleted in alung cancer cell line (Ishikawa et al., Cytogenet Cell Genet1997;78(1):58-60), suggesting an involvement in cancers such as lungcancer. Additionally, a mutation in the pleckstrin homology domain ofPLC-deltal has been found in a patient with early-onset sporadicAlzheimer's diseases (Shimohama et al., Biochem. Biophys. Res. Commun.245:722-728, 1998), and mutations in the pleckstrin homology domain areknown to occur in Bruton agammaglobulinemia (Shimohama et al., Biochem.Biophys. Res. Commun. 245:722-728, 1998). For a further review of PLCproteins, particularly proteins of the PLC-delta subclass, see: Leoniset al., Biochem Biophys Res Commun 1996 Jul 16;224(2):382-90; Cheng etal., J Biol Chem 1995 Mar 10;270(10):5495-505; Suh et al., Cell 1988 Jul15;54(2):161-9; Milting et al., J Muscle Res Cell Motil 1996Feb;17(1):79-84; and Lyu et al., Mammalian Genome 7:501-504, 1996.

Enzymes in the Phospholipase D (“PLD”) family catalyze the hydrolysis ofphosphatidylcholine (“PC”) and other phospholipids to producephosphatidic acid. A range of agonists acting through G protein-coupledreceptors and receptor tyrosine kinases stimulate this hydrolysis.Phosphatidic acid appears to be important as a second messenger capableof activating a diverse range of signaling pathways. PC-specific PLDactivity has been implicated in numerous cellular pathways, includingsignal transduction, membrane trafficking, the regulation of mitosis,regulated secretion, cytoskeletal reorganization, transcriptionalregulation and cell-cycle control. Many proteins are attached to theplasma membrane via a glysylphosphatidylinositol (“GPI”) anchor.Phosphatidylinositol-glycan (“PIG”)-specific PLDs selectively hydrolyzethe inositol phosphate linkage, allowing release of the protein.

Phospholipase proteins, particularly members of the phospholipase Csubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of phospholipase proteins. The present invention advances thestate of the art by providing previously unidentified humanphospholipase proteins that have homology to members of thephospholipase C subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human phospholipase peptides and proteins that arerelated to the phospholipase C subfamily, as well as allelic variantsand other mammalian orthologs thereof. These unique peptide sequences,and nucleic acid sequences that encode these peptides, can be used asmodels for the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate phospholipaseactivity in cells and tissues that express the phospholipase.Experimental data as provided in FIG. 1 indicates expression in humansin pancreas adenocarcinomas, lung, brain anaplastic oligodendrogliomas,placenta choriocarcinomas, pancreas adenocarcinomas, uterus endometrium,colon adenocarcinomas, eye retinoblastomas, and fetal brain.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodesthe phospholipase protein of the present invention. (SEQ ID NO:1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin humans in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, eye retinoblastomas, andfetal brain.

FIG. 2 provides the predicted amino acid sequence of the phospholipaseof the present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding thephospholipase protein of the present invention. (SEQ ID NO:3) Inaddition structure and functional information, such as intron/exonstructure, promoter location, etc., is provided where available,allowing one to readily determine specific uses of inventions based onthis molecular sequence. As illustrated in FIG. 3, SNPs were identifiedat 16 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a phospholipase protein or part of a phospholipaseprotein and are related to the phospholipase C subfamily. Utilizingthese sequences, additional genomic sequences were assembled andtranscript and/or cDNA sequences were isolated and characterized. Basedon this analysis, the present invention provides amino acid sequences ofhuman phospholipase peptides and proteins that are related to thephospholipase C subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these phospholipase peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the phospholipase of the presentinvention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known phospholipase proteins of thephospholipase C subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin pancreas adenocarcinomas, lung, brain anaplastic oligodendrogliomas,placenta choriocarcinomas, pancreas adenocarcinomas, uterus endometrium,colon adenocarcinomas, eye retinoblastomas, and fetal brain. The art hasclearly established the commercial importance of members of this familyof proteins and proteins that have expression patterns similar to thatof the present gene. Some of the more specific features of the peptidesof the present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown phospholipase C family or subfamily of phospholipase proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thephospholipase family of proteins and are related to the phospholipase Csubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the phospholipase peptides of the present invention,phospholipase peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the phospholipase peptides disclosed in the FIG. 2,(encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNAor FIG. 3, genomic sequence), as well as all obvious variants of thesepeptides that are within the art to make and use. Some of these variantsare described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thephospholipase peptide having less than about 30% (by dry weight)chemical precursors or other chemicals, less than about 20% chemicalprecursors or other chemicals, less than about 10% chemical precursorsor other chemicals, or less than about 5% chemical precursors or otherchemicals.

The isolated phospholipase peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, eye retinoblastomas, andfetal brain. For example, a nucleic acid molecule encoding thephospholipase peptide is cloned into an expression vector, theexpression vector introduced into a host cell and the protein expressedin the host cell. The protein can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the phospholipase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The phospholipase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a phospholipase peptideoperatively linked to a heterologous protein having an amino acidsequence not substantially homologous to the phospholipase peptide.“Operatively linked” indicates that the phospholipase peptide and theheterologous protein are fused in-frame. The heterologous protein can befused to the N-terminus or C-terminus of the phospholipase peptide.

In some uses, the fusion protein does not affect the activity of thephospholipase peptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant phospholipase peptide. In certain host cells (e.g.,mammalian host cells), expression and/or secretion of a protein can beincreased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A phospholipase peptide-encoding nucleic acid canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the phospholipase peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the phospholipase peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thephospholipase peptides of the present invention as well as being encodedby the same genetic locus as the phospholipase peptide provided herein.The gene encoding the novel phospholipase protein of the presentinvention is located on a genome component that has been mapped to humanchromosome 17 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data.

Allelic variants of a phospholipase peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the phospholipase peptide aswell as being encoded by the same genetic locus as the phospholipasepeptide provided herein. Genetic locus can readily be determined basedon the genomic information provided in FIG. 3, such as the genomicsequence mapped to the reference human. The gene encoding the novelphospholipase protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 17 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a phospholipase peptideencoding nucleic acid molecule under stringent conditions as more fullydescribed below.

FIG. 3 provides information on SNPs that have been found in the geneencoding the phospholipase proteins of the present invention. SNPs wereidentified at 16 different nucleotide positions. SNPs outside the ORFand in introns may affect control/regulatory elements.

Paralogs of a phospholipase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the phospholipase peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a phospholipase peptide encoding nucleic acid molecule under moderateto stringent conditions as more fully described below.

Orthologs of a phospholipase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the phospholipase peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a phospholipase peptide encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

Non-naturally occurring variants of the phospholipase peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the phospholipase peptide.For example, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a phospholipase peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchangeof the acidic residues Asp and Glu; substitution between the amideresidues Asn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant phospholipase peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as phospholipase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

The present invention further provides fragments of the phospholipasepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a phospholipase peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the phospholipase peptide or could bechosen for the ability to perform a function, e.g. bind a substrate oract as an immunogen. Particularly important fragments are biologicallyactive fragments, peptides that are, for example, about 8 or more aminoacids in length. Such fragments will typically comprise a domain ormotif of the phospholipase peptide, e.g., active site, a transmembranedomain or a substrate-binding domain. Further, possible fragmentsinclude, but are not limited to, domain or motif containing fragments,soluble peptide fragments, and fragments containing immunogenicstructures. Predicted domains and functional sites are readilyidentifiable by computer programs well known and readily available tothose of skill in the art (e.g., PROSITE analysis). The results of onesuch analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in phospholipase peptidesare described in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N. Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the phospholipase peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature phospholipase peptide is fusedwith another compound, such as a compound to increase the half-life ofthe phospholipase peptide (for example, polyethylene glycol), or inwhich the additional amino acids are fused to the mature phospholipasepeptide, such as a leader or secretory sequence or a sequence forpurification of the mature phospholipase peptide or a pro-proteinsequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in aphospholipase-effector protein interaction or phospholipase-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, phospholipases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the phospholipase. Experimental data asprovided in FIG. 1 indicates that phospholipase proteins of the presentinvention are expressed in humans in pancreas adenocarcinomas, lung,brain anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreasadenocarcinomas, uterus endometrium, colon adenocarcinomas, eyeretinoblastomas, and fetal brain. Specifically, a virtual northern blotshows expression in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, and eye retinoblastomas. Inaddition, PCR-based tissue screening panels indicate expression in fetalbrain. A large percentage of pharmaceutical agents are being developedthat modulate the activity of phospholipase proteins, particularlymembers of the phospholipase C subfamily (see Background of theInvention). The structural and functional information provided in theBackground and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in pancreas adenocarcinomas,lung, brain anaplastic oligodendrogliomas, placenta choriocarcinomas,pancreas adenocarcinomas, uterus endometrium, colon adenocarcinomas, eyeretinoblastomas, and fetal brain. Such uses can readily be determinedusing the information provided herein, that which is known in the art,and routine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to phospholipases that are related tomembers of the phospholipase C subfamily. Such assays involve any of theknown phospholipase functions or activities or properties useful fordiagnosis and treatment of phospholipase-related conditions that arespecific for the subfamily of phospholipases that the one of the presentinvention belongs to, particularly in cells and tissues that express thephospholipase. Experimental data as provided in FIG. 1 indicates thatphospholipase proteins of the present invention are expressed in humansin pancreas adenocarcinomas, lung, brain anaplastic oligodendrogliomas,placenta choriocarcinomas, pancreas adenocarcinomas, uterus endometrium,colon adenocarcinomas, eye retinoblastomas, and fetal brain.Specifically, a virtual northern blot shows expression in pancreasadenocarcinomas, lung, brain anaplastic oligodendrogliomas, placentachoriocarcinomas, pancreas adenocarcinomas, uterus endometrium, colonadenocarcinomas, and eye retinoblastomas. In addition, PCR-based tissuescreening panels indicate expression in fetal brain.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the phospholipase, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in pancreas adenocarcinomas, lung, brainanaplastic oligodendrogliomas, placenta choriocarcinomas, pancreasadenocarcinomas, uterus endometrium, colon adenocarcinomas, eyeretinoblastomas, and fetal brain. In an alternate embodiment, cell-basedassays involve recombinant host cells expressing the phospholipaseprotein.

The polypeptides can be used to identify compounds that modulatephospholipase activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with thephospholipase. Both the phospholipases of the present invention andappropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to thephospholipase. These compounds can be further screened against afunctional phospholipase to determine the effect of the compound on thephospholipase activity. Further, these compounds can be tested in animalor invertebrate systems to determine activity/effectiveness. Compoundscan be identified that activate (agonist) or inactivate (antagonist) thephospholipase to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between thephospholipase protein and a molecule that normally interacts with thephospholipase protein, e.g. a substrate or a component of the signalpathway that the phospholipase protein normally interacts (for example,another phospholipase). Such assays typically include the steps ofcombining the phospholipase protein with a candidate compound underconditions that allow the phospholipase protein, or fragment, tointeract with the target molecule, and to detect the formation of acomplex between the protein and the target or to detect the biochemicalconsequence of the interaction with the phospholipase protein and thetarget, such as any of the associated effects of signal transductionsuch as protein phosphorylation, cAMP turnover, and adenylate cyclaseactivation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantphospholipases or appropriate fragments containing mutations that affectphospholipase function and thus compete for substrate. Accordingly, afragment that competes for substrate, for example with a higheraffinity, or a fragment that binds substrate but does not allow release,is encompassed by the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) phospholipase activity.The assays typically involve an assay of events in the signaltransduction pathway that indicate phospholipase activity. Thus, thephosphorylation of a substrate, activation of a protein, a change in theexpression of genes that are up- or down-regulated in response to thephospholipase protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by thephospholipase can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the phospholipase can be assayed.Experimental data as provided in FIG. 1 indicates that phospholipaseproteins of the present invention are expressed in humans in pancreasadenocarcinomas, lung, brain anaplastic oligodendrogliomas, placentachoriocarcinomas, pancreas adenocarcinomas, uterus endometrium, colonadenocarcinomas, eye retinoblastomas, and fetal brain. Specifically, avirtual northern blot shows expression in pancreas adenocarcinomas,lung, brain anaplastic oligodendrogliomas, placenta choriocarcinomas,pancreas adenocarcinomas, uterus endometrium, colon adenocarcinomas, andeye retinoblastomas. In addition, PCR-based tissue screening panelsindicate expression in fetal brain.

Binding and/or activating compounds can also be screened by usingchimeric phospholipase proteins in which the amino terminalextracellular domain, or parts thereof, the entire transmembrane domainor subregions, such as any of the seven transmembrane segments or any ofthe intracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native phospholipase. Accordingly, a different set ofsignal transduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the phospholipase is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the phospholipase (e.g. binding partners and/or ligands). Thus, acompound is exposed to a phospholipase polypeptide under conditions thatallow the compound to bind or to otherwise interact with thepolypeptide. Soluble phospholipase polypeptide is also added to themixture. If the test compound interacts with the soluble phospholipasepolypeptide, it decreases the amount of complex formed or activity fromthe phospholipase target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the phospholipase. Thus, the soluble polypeptide that competes withthe target phospholipase region is designed to contain peptide sequencescorresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the phospholipase protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofphospholipase-binding protein found in the bead fraction quantitatedfrom the gel using standard electrophoretic techniques. For example,either the polypeptide or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin using techniques wellknown in the art. Alternatively, antibodies reactive with the proteinbut which do not interfere with binding of the protein to its targetmolecule can be derivatized to the wells of the plate, and the proteintrapped in the wells by antibody conjugation. Preparations of aphospholipase-binding protein and a candidate compound are incubated inthe phospholipase protein-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the phospholipase protein target molecule, or which arereactive with phospholipase protein and compete with the targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the target molecule.

Agents that modulate one of the phospholipases of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of phospholipase protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the phospholipase pathway, by treating cells ortissues that express the phospholipase. Experimental data as provided inFIG. 1 indicates expression in humans in pancreas adenocarcinomas, lung,brain anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreasadenocarcinomas, uterus endometrium, colon adenocarcinomas, eyeretinoblastomas, and fetal brain. These methods of treatment include thesteps of administering a modulator of phospholipase activity in apharmaceutical composition to a subject in need of such treatment, themodulator being identified as described herein.

In yet another aspect of the invention, the phospholipase proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the phospholipase and are involved inphospholipase activity. Such phospholipase-binding proteins are alsolikely to be involved in the propagation of signals by the phospholipaseproteins or phospholipase targets as, for example, downstream elementsof a phospholipase-mediated signaling pathway. Alternatively, suchphospholipase-binding proteins are likely to be phospholipaseinhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a phospholipaseprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aphospholipase-dependent complex, the DNA-binding and activation domainsof the transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the phospholipase protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a phospholipase-modulating agent, an antisensephospholipase nucleic acid molecule, a phospholipase-specific antibody,or a phospholipase-binding partner) can be used in an animal or othermodel to determine the efficacy, toxicity, or side effects of treatmentwith such an agent. Alternatively, an agent identified as describedherein can be used in an animal or other model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatments as described herein.

The phospholipase proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in pancreas adenocarcinomas, lung, brainanaplastic oligodendrogliomas, placenta choriocarcinomas, pancreasadenocarcinomas, uterus endometrium, colon adenocarcinomas, eyeretinoblastomas, and fetal brain. The method involves contacting abiological sample with a compound capable of interacting with thephospholipase protein such that the interaction can be detected. Such anassay can be provided in a single detection format or a multi-detectionformat such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered phospholipase activity incell-based or cell-free assay, alteration in substrate orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the phospholipase protein in whichone or more of the phospholipase functions in one population isdifferent from those in another population. The peptides thus allow atarget to ascertain a genetic predisposition that can affect treatmentmodality. Thus, in a ligand-based treatment, polymorphism may give riseto amino terminal extracellular domains and/or other substrate-bindingregions that are more or less active in substrate binding, andphospholipase activation. Accordingly, substrate dosage wouldnecessarily be modified to maximize the therapeutic effect within agiven population containing a polymorphism. As an alternative togenotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in humansin pancreas adenocarcinomas, lung, brain anaplastic oligodendrogliomas,placenta choriocarcinomas, pancreas adenocarcinomas, uterus endometrium,colon adenocarcinomas, eye retinoblastomas, and fetal brain.Accordingly, methods for treatment include the use of the phospholipaseprotein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe phospholipase proteins. Antibodies can be prepared from any regionof the peptide as described herein. However, preferred regions willinclude those involved in function/activity and/or phospholipase/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates thatphospholipase proteins of the present invention are expressed in humansin pancreas adenocarcinomas, lung, brain anaplastic oligodendrogliomas,placenta choriocarcinomas, pancreas adenocarcinomas, uterus endometrium,colon adenocarcinomas, eye retinoblastomas, and fetal brain.Specifically, a virtual northern blot shows expression in pancreasadenocarcinomas, lung, brain anaplastic oligodendrogliomas, placentachoriocarcinomas, pancreas adenocarcinomas, uterus endometrium, colonadenocarcinomas, and eye retinoblastomas. In addition, PCR-based tissuescreening panels indicate expression in fetal brain. Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, eye retinoblastomas, andfetal brain. If a disorder is characterized by a specific mutation inthe protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin pancreas adenocarcinomas, lung, brain anaplastic oligodendrogliomas,placenta choriocarcinomas, pancreas adenocarcinomas, uterus endometrium,colon adenocarcinomas, eye retinoblastomas, and fetal brain. Thediagnostic uses can be applied, not only in genetic testing, but also inmonitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in humans in pancreasadenocarcinomas, lung, brain anaplastic oligodendrogliomas, placentachoriocarcinomas, pancreas adenocarcinomas, uterus endometrium, colonadenocarcinomas, eye retinoblastomas, and fetal brain. Thus, where aspecific protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the phospholipase peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornuleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a phospholipase peptide or protein of the present invention(cDNA, transcript and genomic sequence). Such nucleic acid moleculeswill consist of, consist essentially of, or comprise a nucleotidesequence that encodes one of the phospholipase peptides of the presentinvention, an allelic variant thereof, or an ortholog or paralogthereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO: 1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule consists essentially of a nucleotidesequence when such a nucleotide sequence is present with only a fewadditional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the phospholipase peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the phospholipaseproteins of the present invention that are described above. Such nucleicacid molecules may be naturally occurring, such as allelic variants(same locus), paralogs (different locus), and orthologs (differentorganism), or may be constructed by recombinant DNA methods or bychemical synthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel phospholipase protein of the present invention is located on agenome component that has been mapped to human chromosome 17 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

FIG. 3 provides information on SNPs that have been found in the geneencoding the phospholipase proteins of the present invention. SNPs wereidentified at 16 different nucleotide positions. SNPs outside the ORFand in introns may affect control/regulatory elements.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6X sodiumchloride/sodium citrate (SSC) at about 45 C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at 16 different nucleotidepositions.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. The gene encoding the novel phospholipase proteinof the present invention is located on a genome component that has beenmapped to human chromosome 17 (as indicated in FIG. 3), which issupported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates thatphospholipase proteins of the present invention are expressed in humansin pancreas adenocarcinomas, lung, brain anaplastic oligodendrogliomas,placenta choriocarcinomas, pancreas adenocarcinomas, uterus endometrium,colon adenocarcinomas, eye retinoblastomas, and fetal brain.Specifically, a virtual northern blot shows expression in pancreasadenocarcinomas, lung, brain anaplastic oligodendrogliomas, placentachoriocarcinomas, pancreas adenocarcinomas, uterus endometrium, colonadenocarcinomas, and eye retinoblastomas. In addition, PCR-based tissuescreening panels indicate expression in fetal brain. Accordingly, theprobes can be used to detect the presence of, or to determine levels of,a specific nucleic acid molecule in cells, tissues, and in organisms.The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes corresponding to the peptides described herein canbe used to assess expression and/or gene copy number in a given cell,tissue, or organism. These uses are relevant for diagnosis of disordersinvolving an increase or decrease in phospholipase protein expressionrelative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a phospholipase protein, such as bymeasuring a level of a phospholipase-encoding nucleic acid in a sampleof cells from a subject e.g., mRNA or genomic DNA, or determining if aphospholipase gene has been mutated. Experimental data as provided inFIG. 1 indicates that phospholipase proteins of the present inventionare expressed in humans in pancreas adenocarcinomas, lung, brainanaplastic oligodendrogliomas, placenta choriocarcinomas, pancreasadenocarcinomas, uterus endometrium, colon adenocarcinomas, eyeretinoblastomas, and fetal brain. Specifically, a virtual northern blotshows expression in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, and eye retinoblastomas. Inaddition, PCR-based tissue screening panels indicate expression in fetalbrain.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate phospholipase nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe phospholipase gene, particularly biological and pathologicalprocesses that are mediated by the phospholipase in cells and tissuesthat express it. Experimental data as provided in FIG. 1 indicatesexpression in humans in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, eye retinoblastomas, andfetal brain. The method typically includes assaying the ability of thecompound to modulate the expression of the phospholipase nucleic acidand thus identifying a compound that can be used to treat a disordercharacterized by undesired phospholipase nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the phospholipase nucleic acidor recombinant cells genetically engineered to express specific nucleicacid sequences.

The assay for phospholipase nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the phospholipaseprotein signal pathway can also be assayed. In this embodiment theregulatory regions of these genes can be operably linked to a reportergene such as luciferase.

Thus, modulators of phospholipase gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of phospholipasemRNA in the presence of the candidate compound is compared to the levelof expression of phospholipase mRNA in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof nucleic acid expression based on this comparison and be used, forexample to treat a disorder characterized by aberrant nucleic acidexpression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate phospholipase nucleic acid expression incells and tissues that express the phospholipase. Experimental data asprovided in FIG. 1 indicates that phospholipase proteins of the presentinvention are expressed in humans in pancreas adenocarcinomas, lung,brain anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreasadenocarcinomas, uterus endometrium, colon adenocarcinomas, eyeretinoblastomas, and fetal brain. Specifically, a virtual northern blotshows expression in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, and eye retinoblastomas. Inaddition, PCR-based tissue screening panels indicate expression in fetalbrain. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) ornucleic acid expression.

Alternatively, a modulator for phospholipase nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thephospholipase nucleic acid expression in the cells and tissues thatexpress the protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, eye retinoblastomas, andfetal brain.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe phospholipase gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in phospholipase nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in phospholipase genesand gene expression products such as mRNA. The nucleic acid moleculescan be used as hybridization probes to detect naturally occurringgenetic mutations in the phospholipase gene and thereby to determinewhether a subject with the mutation is at risk for a disorder caused bythe mutation. Mutations include deletion, addition, or substitution ofone or more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the phospholipase geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of aphospholipase protein.

Individuals carrying mutations in the phospholipase gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thephospholipase proteins of the present invention. SNPs were identified at16 different nucleotide positions. SNPs outside the ORF and in intronsmay affect control/regulatory elements. The gene encoding the novelphospholipase protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 17 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data. Genomic DNA can be analyzed directly or can beamplified by using PCR prior to analysis. RNA or cDNA can be used in thesame way. In some uses, detection of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al., NucleicAcids Res. 23:675-682 (1995)). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

Alternatively, mutations in a phospholipase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantphospholipase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995) iBiotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly. the nucleic acid molecules described hereincan be used to assess the mutation content of the phospholipase gene inan individual in order to select an appropriate compound or dosageregimen for treatment. FIG. 3 provides information on SNPs that havebeen found in the gene encoding the phospholipase proteins of thepresent invention. SNPs were identified at 16 different nucleotidepositions. SNPs outside the ORF and in introns may affectcontrol/regulatory elements.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol phospholipase gene expression in cells, tissues, and organisms.A DNA antisense nucleic acid molecule is designed to be complementary toa region of the gene involved in transcription, preventing transcriptionand hence production of phospholipase protein. An antisense RNA or DNAnucleic acid molecule would hybridize to the mRNA and thus blocktranslation of mRNA into phospholipase protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of phospholipase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired phospholipase nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the mRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the phospholipaseprotein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in phospholipase geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredphospholipase protein to treat the individual.

The invention also encompasses kits for detecting the presence of aphospholipase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that phospholipase proteins of the presentinvention are expressed in humans in pancreas adenocarcinomas, lung,brain anaplastic oligodendrogliomas, placenta choriocarcinomas, pancreasadenocarcinomas, uterus endometrium, colon adenocarcinomas, eyeretinoblastomas, and fetal brain. Specifically, a virtual northern blotshows expression in pancreas adenocarcinomas, lung, brain anaplasticoligodendrogliomas, placenta choriocarcinomas, pancreas adenocarcinomas,uterus endometrium, colon adenocarcinomas, and eye retinoblastomas. Inaddition, PCR-based tissue screening panels indicate expression in fetalbrain. For example, the kit can comprise reagents such as a labeled orlabelable nucleic acid or agent capable of detecting phospholipasenucleic acid in a biological sample; means for determining the amount ofphospholipase nucleic acid in the sample; and means for comparing theamount of phospholipase nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect phospholipaseprotein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application W095/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14:1675-1680) and Schena, M.et al. (1996; Proc. Natl. Acad. Sci. 93:10614-10619), all of which areincorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application W095/251 116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the phospholipase proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of thephospholipase gene of the present invention. FIG. 3 provides informationon SNPs that have been found in the gene encoding the phospholipaseproteins of the present invention. SNPs were identified at 16 differentnucleotide positions. SNPs outside the ORF and in introns may affectcontrol/regulatory elements.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985), Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified phospholipase gene of the present invention canbe routinely identified using the sequence information disclosed hereincan be readily incorporated into one of the established kit formatswhich are well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E coli, Streptomyces, and Salmonella typhimurium. Eukaryoticcells include, but are not limited to, yeast, insect cells such asDrosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterophospholipase. Typical fusion expression vectorsinclude pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New EnglandBiolabs, Beverly, Me.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Examples ofsuitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology. Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSecl (Baldari, et al, EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such asphospholipases, appropriate secretion signals are incorporated into thevector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with phospholipases, the protein can be isolated from the hostcell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing aphospholipase protein or peptide that can be further purified to producedesired amounts of phospholipase protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe phospholipase protein or phospholipase protein fragments, such asthose described above as well as other formats known in the art. Thus, arecombinant host cell expressing a native phospholipase protein isuseful for assaying compounds that stimulate or inhibit phospholipaseprotein function.

Host cells are also useful for identifying phospholipase protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantphospholipase protein (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native phospholipaseprotein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a phospholipaseprotein and identifying and evaluating modulators of phospholipaseprotein activity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the phospholipase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the phospholipase protein toparticular cells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, phospholipase protein activation, and signal transduction, maynot be evident from in vitro cell-free or cell-based assays.Accordingly, it is useful to provide non-human transgenic animals toassay in vivo phospholipase protein function, including substrateinteraction, the effect of specific mutant phospholipase proteins onphospholipase protein function and substrate interaction, and the effectof chimeric phospholipase proteins. It is also possible to assess theeffect of null mutations, that is, mutations that substantially orcompletely eliminate one or more phospholipase protein functions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

What is claimed is:
 1. An isolated nucleic acid molecule encoding aphospholipase C protein consisting of a nucleotide sequence selectedfrom the group consisting of: (a) a nucleotide sequence that encodes aprotein comprising the an amino acid sequence of SEQ ID NO:2; (b) anucleic acid molecule consisting of the nucleic acid sequence of SEQ. IDNO:1; and (c) a nucleic acid molecule consisting of the nucleic sequenceof SEQ. ID NO:3.
 2. A nucleic acid vector comprising a nucleic acidmolecule of claim
 1. 3. A host cell containing the vector of claim
 2. 4.A process for producing a polypeptide comprising culturing the host cellof claim 3 under conditions sufficient for the production of saidpolypeptide, and recovering the peptide from the host cell culture. 5.An isolated polynucleotide consisting of a nucleotide sequence set forthin SEQ ID NO:1.
 6. An isolated polynucleotide consisting of a nucleotidesequence set forth in SEQ ID NO:3.
 7. A vector according to claim 2,wherein said vector is selected from the group consisting of a plasmid,virus, and bacteriophage.
 8. A vector according to claim 2, wherein saidisolated nucleic acid molecule is inserted into said vector in properorientation and correct reading frame such that the protein of SEQ IDNO:2 may be expressed by a cell transformed with said vector.
 9. Avector according to claim 8, wherein said isolated nucleic acid moleculeis operatively linked to a promoter sequence.
 10. An isolated nucleicacid molecule consisting of a nucleotide sequence that is completelycomplementary to a nucleotide sequence of claim 1.